Effect of hypoxia of adhesion and signaling proteins in the wounded corneal epithelium
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As the outermost layer of the eye, the corneal epithelium is exposed to a wide variety of environmental stressors. Many researchers have studied its response to stressful conditions such as hypoxia and abrasion, but less work has been done in scenarios where these conditions occur together. In order for the cornea to withstand these stressors and return to its original architecture that is critical for proper vision, tightly regulated signaling cascades are necessary to control cell migration and wound repair of the corneal epithelium. It has been established that hypoxia impairs wound healing in the cornea. Here, we carefully investigate individual proteins involved in the corneal epithelium's response to wounding, and how hypoxic conditions affect their expression and localization. We examine the adhesion proteins known to affect epithelial integrity, such as paxillin, E-cadherin, and f34 integrin, to further explain the delayed wound healing in hypoxia. Furthermore, factors such as glutamate and nucleotides are important to mediating the wound healing response. We also examine proteins of the glutamatergic and purinergic receptor families to elucidate the effect of hypoxia on the cell signaling controlling proper cell migration in wound healing, and to further the work done by our lab on the contribution of neuronal factors released during wounding to cell migration. Using immunohistochemical techniques and confocal imaging on cell culture and organ culture, we are able to observe the alterations over time in both two-dimensional and three-dimensional models of wound healing. Our results revealed alterations in expression of adhesion proteins, and an upregulation of purinergic signaling proteins in hypoxic conditions. We also observed more cytosolic P2X7 but decreased NMDAR-1 in extended hypoxia in wounded and unwounded HCLEs. Our studies on neuronal factors showed an upregulation in NMDAR-1 and pannexin in addition to increased directionality of cell migration when exposed to neuronal wound media. Altogether, our results illustrate the importance of normal oxygen levels and neuronal signaling factors in proper wound healing.
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